Lab 23: PCR and Gel Electrophoresis

Question 1

what is the purpose of PCR?

Answer 1

to amplifiy target DNA:
to create billions of copies of a specific region of DNA called the target DNA

Question 2

what is PCR testing used for?

Answer 2

disease diagnosis
species identification
amplify DNA for cloning processes

Question 3

what are the 3 steps of PCR?

Answer 3

1. Denaturation
2. Annealing
3. Synthesis/Extension

Question 4

what happens in denaturation?

Answer 4

-Specificity: to design primers complementary to the ends of target DNA sequence
-Denaturation: unzips the DS DNA by heating at 94 degrees Celsius for 1 minute

Question 5

what happens in annealing?

Answer 5

Cooling down - which allows the primers to anneal or bind to the complementary sequences on target DNA - which is at 60 degrees Celsius for 1 minutes

Question 6

what happens in synthesis/extension?

Answer 6

-The target DNA is copied by a thermostable DNA polymerase called TAQ - this is done at 72 degrees Celsius for 2 minutes.
-This uses the process of DNA replication

Question 7

what do we get after steps 1-3? what do we do after?

Answer 7

We get 2 DS DNA molecules.
Then, we repeat steps 1-3 for 40 times

Question 8

what is the result after this whole process including the 40 times repeated?

Answer 8

billions of target DNA copies

Question 9

what are the components needed for a PCR reaction?

Answer 9

-Template DNA
-Primers for target DNA
-Taq DNA polymerase
-Nucleotides (dNTPs)
-Buffer

Question 10

what is Taq DNA polymerase important for?

Answer 10

Because it is thermostable, it doesn’t denature at high temperatures from step 1

Question 11

what is gel electrophoresis used for?

Answer 11

to detect PCR products

Question 12

what is the gel that we pour?

Answer 12

We pour an agarose (polysaccharide) gel, which has a matrix-like structure with small pores through which DNA molecules can travel

Question 13

what happens after we pour the gel?

Answer 13

we apply an electrical current - the DNA molecules run towards red (the positive electrode) as they are negatively charged

Question 14

what size DNA molecules move faster through the gel?

Answer 14

smaller DNA molecules - separation based on size

Question 15

what is the loading dye?

Answer 15

it is a dye that helps monitor the movement of samples; switch off electrical current when dye gets to the bottom

Question 16

what is the molecular weight ladder?

Answer 16

a standard consisting of a mixture of DNA fragments with known sizes - it helps determine the size of an unknown DNA

Question 17

what is SYBR green?

Answer 17

a dye that binds to the DNA allowing detection - it fluoresces when exposed to UV light

Question 18

what was the goal of the lab?

Answer 18

to determine which one (A, B, or C) has Streptococcus pneumoniae 19A?

Question 19

So we took the sputum samples from 3 patients - what did we do to the samples?

Answer 19

-grow cultures
-isolate DNA
-run PCR with both primer pairs
-compare with a (+) control of S. pneumoniae 19A to see which patient is infected with that strain

Question 20

what two primers did we use?

Answer 20

Primer 1: to detect a gene present in all S. pneumoniae (all pt’s tested have S. pneumoniae gene).

Primer 2: to detect a gene present in only S. pneumoniae 19A strain (infected pt only has this S. pneumoniae 19A strain)

Question 21

what does the control contain in the lab?

Answer 21

The control has BOTH primers, 1 and 2. So it has S. pneumoniae and S. pneumoniae 19A.

Question 22

what are we trying to find out in the lab?

Answer 22

We are trying to see which pt has the S. pneumoniae 19A gene. AKA which pt has both bands from the C+.

Question 23

slide 13

Question 24

T/F: we can’t visualize the dye until it is under the UV

Answer 24

True